JPH0558808A - Flower budding induction agent - Google Patents

Abstract

PURPOSE:To obtain a flower budding induction agent capable of readily modulat ing flowering period regardless of the condition of day length because of its excellent flower budding induction activity. CONSTITUTION:A flower budding induction agent containing one or more kinds of 18C trihydroxyunsaturated fatty acids contained in Lemna paucicostata Hegelm and expressed by formula I [either one of R<1> and R<2> is CxHy and the other is formula II; (x) is integer of 2-10; (y) is 2x+1 or 2x-1; (z) is (12-x) or 2 (12-x)-2; preferably either one of R<1> and R<2> is C5H11 or C5H9 and the other is C7H14COOH] such as a compound expressed by formula III as an active ingredient, capable of exhibiting excellent flower budding induction activity to Lemna paucicostate Hegelm as well as other plants, especially to a plant capable of controlling its budding by day length. The activity of this agent is enhanced by combinedly using norepinephrine (NE). As amount of these ingredients used, the compound expressed by formula I is preferably contained in a ratio of 1-100ppm, especially 10-100ppm and NE is preferably contained in a ratio of about 10muM (1-2ppm) based on 1-500ppm compound expressed by formula I.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a C18-trihydroxyunsaturated fatty acid having a specific structure, or a novel flower bud formation-inducing agent containing the trihydroxyunsaturated fatty acid and norepinephrine. ..

[0002]

2. Description of the Related Art Generally, the flowering time of higher plants is seasonally constant depending on its type, and this is often determined by the length of daily sunshine. For example, as a "short day plant" that blooms when sunshine hours are short, rice,
Maize, chrysanthemum, morning glory, etc. are known, and conversely, barley, wheat, pea, etc. are known as "long-day plants" that bloom when sunshine hours are long. Utilizing this, for example, for chrysanthemums in autumn, artificially controlling the sunshine duration to adjust the flowering time is used, but this is due to the use of special facilities and cultivation areas. There is a problem of cost due to the requirement for movement. Therefore, studies to control the flowering time by the addition of a drug instead of the duration of sunshine, that is, the daylength condition have been actively conducted. It is expected that the regulation by this drug will be an extremely important technique for agriculture not only facilitating the control of flowering time, but also controlling the time of harvest of agricultural products and the number of times of harvest. ing.

In "flowering", there are stages of flower bud differentiation and stages of flower bud development after flower bud differentiation. In the present specification, the former is hereinafter referred to as "flower bud formation" and the latter is referred to as "flower bud formation". Flowering ". It has been suggested that a flower bud formation-inducing substance (flowering hormone) is present in all plants, but extraction of this substance has not been successful yet. However, substances exhibiting flower bud formation-inducing activity are known for some plants. For example, gibberellin, which is a plant growth hormone, promotes flower bud formation in conifers such as cedar, and Ethephon, which is an ethylene generator, is ananas. It has been put to practical use to promote flowering of plants. However, all of them have problems such as different reactions depending on the type of plant. Despite the high interest mentioned above, their use is in a very limited range ["Full scope of plant industry", C Co., Ltd. Published by MC, pp.195-238 (1986
-4-25), and "Bioassay for physiologically active substances", Kodansha Co., Ltd., pp. 151 (1984-2-20)]. Further, as described above, since the main factor that regulates flower bud formation and flowering of plants is photoperiod, a regulator that acts on flower bud formation and flowering due to photoperiod is most desired. Currently, the development of such drugs is not so advanced.

The trihydroxy unsaturated fatty acid having 18 carbon atoms represented by the general formula (1), which is the active ingredient of the present invention, is a compound represented by the formulas (1) -A to (1) -F. Found in seed plants.

[0005]

[Chemical 3]

[0006]

[Chemical 4]

[0007]

[Chemical 5]

[0008]

[Chemical 6]

[0009]

[Chemical 7]

[0010]

[Chemical 8]

For example, M. Takasugi et al. From the root of common bean [Chem. Lett., Pp. 947-950 (1974)], JH Cardel.
linaII et al. from a kind of cyanobacteria (Lyngbya majuscula) [Te
trahedron, 36, pp.993-996 (1980)], W. Herz et al. from several Asteraceae plants [eg Phytochemistry, 24 (1), p.
p.89-91 (1985)] isolated. There is also a report that it was isolated from rice with blast disease and confirmed by chemical synthesis [T. Kato et al .; Tetrahedron Lett., 26 (19), pp.2356.
-2360 (1985), H. Suemune et al .; Chem. Pharm. Bull., 36.
(9), pp.3632-3637 (1988)]. In addition, there are reports that it has various physiologically active actions. For example, Panosyan, AG et al. Found that they have prostaglandin activity, isolated from a species of the Cucurbitaceae (Bryonia alba) [Chemical A
bstract90 (19): 148458j], Shirinyan, EA, et al., isolated from licorice as an anti-stress substance [Chemical Abstr
act 110 (13): 108136c], H. Masui et al. have isolated it from taro as an antifungal substance [Phytochemistry, 28 (1
0), pp.2613-2615 (1989)]. However, there is no report that it has a flower bud formation or flowering regulation effect. Further, the present inventors extracted some of the above-mentioned trihydroxyunsaturated fatty acids having 18 carbon atoms from a duckweed plant, but there is no such report.

[0012] Lemnaceae plants are smaller than other plants, have a faster growth rate, are mostly short-day plants, and can easily control flower bud formation by changing the day length. Because of its properties, it has long been used as an assay system for searching for flower bud formation regulators. As an unsaturated fatty acid among the components of the duckweed plant, it was confirmed that the leaves of duckweed (Spirodela polyrhiza) contained a considerable amount of linolenic acid and linoleic acid [D. Lechevallier; Chemical Abstract 66
(15): 62625d], and also Lemna duckweed (Lemn
a minor) and 10-hydroxyhexadeca-7,11,13-trienoic acid with 16 carbon atoms was isolated [L.Previt
era et al .; Phytochemistry, 22 (6), pp.1445-1446 (198
3)], 12-hydroxyhexadeca-8,10,14-trienoic acid has been isolated from the same genus, L. trisulca [P. Monaco et al .; Phytochemistry, 26 (3), pp.
745-747 (1987)].

However, no report has been made on the relationship between unsaturated fatty acids contained in these duckweed plants and flower bud formation. Benzoic acid, nicotinic acid, nicotinamide, pipecolic acid, etc. have been found as a flower bud formation inducer in duckweed plants from acetone extracts of duckweed plants [S. Fujioka et al .; Plant Cell
Physiol., 28 (6), pp.995-1003 (1987)]. Furthermore, in the present invention, norepinephrine (hereinafter abbreviated as “NE”) added to the above unsaturated fatty acids is one of catecholamines known as a human chemical mediator, but is cultured under short-day conditions. It has been reported that flowering is promoted when it is supplied to the strain 6746 of the duckweed L. paucicostata (JP Khur).
ana et al .; Plant Physiol., 85 (1), pp.10-12 (1987).
]. Takimoto et al., Who is one of the present inventors, found that this NE is contained in duckweed itself and that NE alone does not show flower bud formation-inducing activity for duckweed, but a precipitate obtained by centrifugation of a water extract of duckweed It was found that the combination of these compounds shows activity, and we have already reported [Plan
t Cell Physiol., 32 (2), pp.283-289 (1991)].

[0014]

DISCLOSURE OF THE INVENTION The present invention is based on the elucidation of a flower bud formation-inducing substance in duckweed and is an excellent flower bud formation induction which facilitates the control of flowering time regardless of the day length condition. The purpose is to provide an agent.

[0015]

[Means for Solving the Problems] In view of the above-mentioned problems, the inventors of the present invention have conducted diligent research on a flower bud formation-inducing substance in duckweed, and as a result, have a carbon number of 18 having a specific structure contained in duckweed. It was found that trihydroxy unsaturated fatty acid shows excellent flower bud formation-inducing activity for duckweed itself, and further shows excellent activity for other plants, especially plants whose flower bud formation is regulated by photoperiod,
Further, they have found that the activity is enhanced when used in combination with NE, and completed the present invention. That is, the present invention has the general formula (1)

[0016]

[Chemical 9]

[Wherein ^one of R ¹ and R ² is C _x H _y
(X means an integer of 2 to 10 and y is 2x + 1 or 2
It means an integer represented by x-1. ) Means an alkyl group or an alkenyl group, and the other is C _(12-x)
H _z COOH (x means the same as above, z is 2
It means an integer represented by (12-x) or 2 (12-x) -2. ) Means a ω-hydroxycarbonyl-substituted alkyl group or a ω-hydroxycarbonyl-substituted alkenyl group. ] A flower bud formation-inducing agent containing one or more trihydroxyunsaturated fatty acids having 18 carbon atoms represented by the formula, and the trihydroxyunsaturated fatty acids and NE. It is a flower bud formation inducer.

The C18-trihydroxyunsaturated fatty acid represented by the general formula (1), which is the active ingredient of the present invention (hereinafter abbreviated as "fatty acid (1)"), is described in the section of the prior art. Although the plants described above can be obtained as a raw material, they can be easily obtained by the method first isolated from duckweed by the present inventors, for example, by the extraction and purification means shown below.

That is, water is added to the duckweed to grind it, and the mixture is left to stir at room temperature for about 5 to 12 hours and then centrifuged, and the resulting supernatant is collected at about 90 to 100 ° C. for 5 to 10 hours.
The unnecessary substances such as the enzyme contained are treated by heating for 1 minute, cooled, and then centrifuged again to extract the supernatant obtained with an organic solvent. Here, as the organic solvent used, ethyl acetate, diethyl ether, n-butanol,
Chloroform, dichloromethane, tetrahydrofuran and the like can be mentioned, and among them, ethyl acetate is preferable because it allows efficient extraction. Then, the oil obtained by distilling off the extraction solvent is subjected to silica gel column chromatography to obtain an appropriate developing solvent, for example, chloroform, methanol, benzene, a mixed solvent thereof, or a mixed solvent thereof, and further acetic acid is mixed with the mixed solvent. The target fatty acid (1) can be obtained in the form of a mixture by elution with the above solvent or the like. Although this mixture can be used as it is as an active ingredient of the agent for inducing flower bud formation of the present invention, high performance liquid chromatography using a column for reverse phase partition chromatography such as a column for gel permeation chromatography (GPC) or an ODS column. (HPL
It is also possible to separate each compound by C).

The fatty acid (1) can also be easily obtained by a synthetic reaction. For example, T. Kato et al .; Tetrahedron Lett., 26 (19), pp.23 described in the section of the prior art.
56-2360 (1985) or H. Suemune et al .; Chem. Pharm. Bull.,
According to the method described in 36 (9), pp.3632-3637 (1988), dimethyl tartrate can be obtained as a starting material. The fatty acid (1) thus obtained has an excellent flower bud formation-inducing activity, and as the active ingredient of the flower bud formation-inducing agent of the present invention, only one kind may be blended, or two or more kinds may be mixed. May be.

Specific production examples of the fatty acid (1) which is the active ingredient of the present invention and test examples of its flower bud formation inducing activity are shown below.

Production Example A strain 441 of Lemna paucicostata (hereinafter referred to as "Lemna paucicostata") cultured under continuous light with Hyponex (trade name, liquid fertilizer manufactured by Murakami Bussan Co., Ltd.) diluted to 1/1000.
It is abbreviated as "P441". ) 2.0 kg (fresh weight) was crushed by adding the same weight of water, and left stirring at room temperature for 6 hours. The supernatant obtained by centrifugation at 12000 × g for 30 minutes was heat-treated at 90 ° C. for 5 minutes, cooled, and then centrifuged again. The supernatant was extracted twice with 1.5 liters of ethyl acetate, the solvent was distilled off, and 0.8 g of the obtained oil was subjected to silica gel column chromatography to obtain chloroform: methanol = 7: 1 (volume ratio, Elute with a mixed solvent of (the same) and confirm by thin layer chromatography that R
The f (rate of flow) migration rate of 0.15 part was fractionated. Furthermore, 0.2 g of the obtained aliquot was subjected to silica gel column chromatography to obtain benzene: methanol: acetic acid =
The Rf 0.3 portion was separated by elution with a mixed solvent of 45: 8: 2 to obtain 0.01 g of a desired mixture of fatty acids (1) (hereinafter abbreviated as "fatty acid group") (yellow). Oil).

Then, HPLC using an ODS column
Then, using 30% acetonitrile aqueous solution containing 0.05% trifluoroacetic acid as a mobile phase, three different fatty acids (1) -A, (1) -B and (1) -C shown below are used.
Was obtained (1.0 mg, 1.2 mg, 1.0 mg, respectively) (all white oils). These structures were confirmed by measuring a nuclear magnetic resonance spectrum ( ¹ H-NMR) and a mass spectrum.

[0024]

[Chemical 10]

[0025]

[Chemical 11]

[0026]

[Chemical formula 12]

Test Example 1 Based on the method described in "Bioassay of physiologically active substances", published by Kodansha, Ltd., p.152-155 (1984-2-20), the active ingredient obtained in the above Production Example was tested for duckweed. System 1
The flower bud formation-inducing activity for 51 (hereinafter abbreviated as "P151") was examined.

As the test compound (group), the fatty acid group obtained in Production Example, trihydroxy unsaturated fatty acid (1) -A,
Prepared by dissolving 1 mg of each of (1) -B and (1) -C in 1 ml of a 1% aqueous acetonitrile solution,
1/10 diluted E culture medium to which 1 μM (micromol / liter) of benzyladenine (hereinafter abbreviated as “BAd”) was added so that the concentration of each test compound becomes the concentration shown in Table 2 below. (The composition is shown in Table 1.) 10
The solution was added to an Erlenmeyer flask containing milliliters (hereinafter, abbreviated as "NE-free flask"). Similarly, the test compound (group) and the compound of formula (2) as a comparative compound (a trihydroxy unsaturated fatty acid having a structure similar to that of the active ingredient of the present invention, the positions of the hydroxyl group and the double bond are Differently, 1 mg of each of the above-mentioned production examples separated from a portion other than Rf0.3) was dissolved in 1 ml of a 5% acetonitrile solution so that the concentration of the test compound becomes the concentration shown in Table 2 below. To which was added 10 μM NE and 1 μM BAd 1
/ 10 diluted E culture solution was added to an Erlenmeyer flask (hereinafter abbreviated as “flask with NE”) containing 10 ml.

[0029]

[Chemical 13]

Into the flask without NE and the flask with NE, 3 frond colonies (leaflets) of P151 were inoculated and cultured at 25 ° C. under continuous light of 5000 lux for 7 days to examine whether flower buds were formed. The flower bud formation rate was determined based on the method described in the above document, and the results are shown in Table 2.
In addition, a flask without NE and a flask with NE under exactly the same conditions except that the test compound (group) was not added was used as a control.

[0031]

[Table 1] Table 1 E Composition of culture solution ───────────────────────────────────── Composition Amount (mg / liter) ──────────────────────────────────── KH ₂ PO ₄ 68000 KNO ₃ 1515 Ca (NO ₃ ) ₂ .4H ₂ O 1180 MgSO ₄ .7H ₂ O 492 H ₃ BO ₃ 2.86 ZnSO ₄ .7H ₂ O 0.22 Na ₂ MoO ₄ .2H ₂ O 0.12 MnCl ₂ .4H _{2 O 3.62 FeCl 2 · 6H 2 O} 5.40 tartrate 3.0 disodium ethylenediaminetetraacetate 30 [mu] M ─────────────────────────── ───────── pH 4.6 ─────────────────────────────────────

[0032]

[Table 2]

As described in the above-mentioned "Bioassay of physiologically active substances", Kodansha, Ltd., p.152-155 (1984-2-20), in the case of duckweed, the flower bud formation rate is 100%. Rarely, it is judged that when it is 50% or more, a very strong induction of flower bud formation is received. Therefore, the fatty acid (1) which is the active ingredient of the present invention is P151.
On the other hand, it is clear that when NE is added, a particularly strong flower bud formation-inducing activity is exhibited.

Test Example 2 J. Ullmann et al .; Biologia Plantarum 27 (4-5), pp.367-3
72 (1985), based on the fatty acid group obtained in the above production example, Chenopod, which is a plant of the genus Acatha
The flower bud formation-inducing activity against ium rubrum (hereinafter abbreviated as "rubrum") was examined. That is, 0.5 g of rubrum seeds was sown under illumination at 30 ° C., allowed to stand for 20 hours and then subjected to dark treatment at 10 ° C. for 12 hours to obtain a uniform germination state. This was hydroponically cultivated in a petri dish containing 4 ml of ½ diluted Knop medium (composition is shown in Table 3), and 20
It was cultivated for 12 days under continuous light of 8000 lux at ℃.
Dark treatment was performed for 12 hours before treatment with the test compound group. Treatment with the test compound group was carried out once by a method of placing 3 microliters of a 1000 ppm aqueous solution of fatty acid group-1 at the vegetative growth point and a method of absorbing 1 ml of the same aqueous solution from the roots, and after 5 days, it was grown by a microscope. The formation of flower buds was examined by observing the morphological change of the spots, and the flower bud formation rate was determined to be 0% in the control which was not treated with the test compound, whereas it was 100 in the growth point. %, And 75% when absorbed from the roots.

[0035]

[Table 3] Table 3 Composition of Knop medium ───────────────────────────────────── Composition amount (mg / _{l) ──────────────────────────────────── MgSO 4 · 7H 2 O} 250 KNO 3 _{250 Ca (NO 3) 2 ·} 4H 2 O 1000 KH 2 PO 4 250 ───────────────────────────────── ───

To prepare the flower bud formation inducer of the present invention,
The above-mentioned active ingredient, fatty acid (1) or fatty acid (1) and NE, can be used as a carrier such as a known solid carrier or liquid carrier such as talc, clay, vermiculite, diatomaceous earth, kaolin, calcium carbonate, calcium hydroxide. , Clay, inorganic substances such as silica gel, solid carriers such as wheat flour and starch, or water; aromatic hydrocarbons such as xylene;
Paraffin hydrocarbons such as mineral oil; alcohols such as ethanol and ethylene glycol; ketones such as acetone; ethers such as dioxane and tetrahydrofuran; diluted with a liquid carrier such as dimethylformamide, dimethylsulfoxide, acetonitrile, etc. , Other pharmaceutical auxiliaries such as surfactants, if necessary, eg alkyl sulfates, alkyl sulfonates,
Anionic surfactants such as alkylaryl sulfonates and dialkyl sulfosuccinates; cationic surfactants such as salts of higher aliphatic amines; polyoxyethylene glycol alkyl ethers, polyoxyethylene glycol acyl esters, polyoxy Nonionic surfactants such as ethylene glycol polyhydric alcohol acyl esters and cellulose derivatives; powders, granules, emulsions, suspensions, wettable powders, water solutions, liquids by adding gelatin, casein, gum arabic, etc. Etc. can be prepared. The active ingredient may be added to the fertilizer as it is or as a formulation. Furthermore, it is also possible to mix and use other components such as a plant growth regulator. For example,
Benzoic acid, nicotinic acid, nicotinamide, pipecolic acid, etc. described in the section of the conventional technique may be added as necessary.

The flower bud formation-inducing agent of the present invention thus obtained is applied by a method such as spraying, dripping, coating on the vegetative growth point of a plant, or absorbing from a root. The amount used varies depending on the type of the target plant, growth conditions, application method, etc., but usually the active ingredient fatty acid (1) is 1 to 1
It is preferable to set it to 000 ppm, preferably 10 to 100 ppm. Moreover, when NE is contained, about 10 micromol (1-
2 ppm) is preferable.

[0038]

[Examples] Examples of preparations of the flower bud formation-inducing agent of the present invention are shown below as examples, but the present invention is not limited thereto. Example 1 Dust Preparation Fatty acid group obtained in Production Example 0.1 parts by weight Talc 20 parts by weight Clay 80 parts by weight The above components were mixed to give a powder.

Example 2 Dusts NE was added to the formulation of Example 1 so as to be 3.5 ppm and mixed to obtain a dust.

Example 3 Emulsion Fatty acid group obtained in Production Example 1 part by weight Polysorbate 80 2 parts by weight The above components were mixed and appropriately diluted with water, and NE was further adjusted to 10 μm.
An emulsion was prepared by adding M so that M was obtained.

Example 4 Liquid formulation Fatty acid group obtained in Production Example 1 part by weight 5% acetonitrile aqueous solution 50 parts by weight The above components were mixed to prepare a liquid formulation. Similarly, instead of the fatty acid group, a liquid preparation containing the fatty acids (1) -A, (1) -B and (1) -C obtained in Production Example was prepared. In use, this is appropriately diluted with water, and NE is further added to 10 μM to spray.

[0042]

INDUSTRIAL APPLICABILITY The flower bud formation-inducing agent of the present invention contains a trihydroxyunsaturated fatty acid having a specific structure and having 18 carbon atoms, or the trihydroxyunsaturated fatty acid and norepinephrine and has an excellent flower bud formation-inducing activity Therefore, it is possible to easily control the flowering time regardless of the day length condition.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Atsushi Takimoto 50 Shishikuueshibahonmachi, Kita-ku, Kyoto-shi, Kyoto Prefecture

Claims (4)

[Claims] 1. A compound represented by the general formula (1): [In the formula, ^one of R ¹ and R ² is represented by C _x H _y (x represents an integer of 2 to 10, and y represents an integer represented by 2x + 1 or 2x-1). means an alkyl or alkenyl group, the _{other, C (12-x) H} z COOH
(X means the same as above, z means an integer represented by 2 (12-x) or 2 (12-x) -2), or a ω-hydroxycarbonyl-substituted alkyl group or It means an ω-hydroxycarbonyl-substituted alkenyl group. ] The flower bud formation inducer characterized by containing 1 type, or 2 or more types of C18 trihydroxy unsaturated fatty acid represented by these. 2. The trihydroxy unsaturated fatty acid according to the general formula (1), wherein one of R ¹ and R ² is C ₅ H ₁₁ or C ₅ H ₉ and the other is C ₇ H ₁₄ COOH. The described flower bud formation inducer. 3. A compound represented by the general formula (1): [In the formula, ^one of R ¹ and R ² is represented by C _x H _y (x represents an integer of 2 to 10, and y represents an integer represented by 2x + 1 or 2x-1). means an alkyl or alkenyl group, the _{other, C (12-x) H} z COOH
(X means the same as above, z means an integer represented by 2 (12-x) or 2 (12-x) -2), or a ω-hydroxycarbonyl-substituted alkyl group or and a norepinephrine at least one trihydroxy unsaturated fatty acid having 18 carbon atoms and norepinephrine. 4. The trihydroxy unsaturated fatty acid according to the general formula (1), wherein one of R ¹ and R ² is C ₅ H ₁₁ or C ₅ H ₉ and the other is C ₇ H ₁₄ COOH. The described flower bud formation inducer.